Project Summary Bleomycin (BLM) treatment is a standard, but imperfect, mouse model that is used by countless investigators to study acute lung injury (ALI) and pulmonary fibrosis. One of the major difficulties in using the BLM model is the variability in results from lab to lab. As the technical aspects of the model are straight forward, why there is inconsistency in the results remains mysterious. Understanding the mechanisms behind these disparate results in a mouse model could provide valuable clues to human disease such as ALI and pulmonary fibrosis. Why some patients die of these diseases and others recover remains an important and unanswered question. One potential clue may relate to the microbiome, required for normal immune development and of a normal host response in the lung. Recent studies suggest that fibrotic lung disease may be associated with an altered microbiome. Hence, there is a critical link between the host's inherent microbes and lung injury associated with fibrotic lung disease, yet the association between the host microbiome and susceptibility to these diseases has not been explored. We have exciting preliminary data that the microbiome plays a critical role in survival in the BLM mouse model of ALI. We have found that the wild-type (WT) littermate controls of multiple strains on the C56Bl/6 (B6) background have completely different responses after BLM treatment depending in which mouse facility they are housed. Mice housed in Facility A had minimal weight loss and mostly survived, while mice housed in Facility B, across the street, had severe weight loss and mostly perished. These dramatic differences occurred despite both facilities both being Specific Pathogen Free (SPF), and both using laminar flow ventilated racks for housing. These data suggested a role for the microbiota in the disparate responses to BLM. To directly address whether the microbial environment could drive these differences, littermate germ-free (GF) male mice were relocated and conventionalized in either Facility A or B. The GF mice that were conventionalized in Facility A lost less weight and had improved survival compared to the mice conventionalized in Facility B: this confirmed a role for housing environment and microbiota. Additional data generated from 16s rRNA amplicon sequence analysis of fecal samples taken from these mice demonstrated significant differences in alpha- and beta-diversity, and in microbial taxonomy, indicating that the gut microbiomes in mice housed in Facility A and B are both clearly distinct and clearly can direct the disease course and recovery from acute lung injury. Our long-term goal is to identify specific microbes that improve or worsen the response to ALI so that patient care can be tailored to promote a protective microbiome. We hypothesize that specific gut and/or lung microbial communities alter the immunologic and fibrotic response to BLM-induced lung injury.